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Dynamic regulation of transcription complex recruitment to the β-globin gene locus
ABSTRACTS / Blood Cells, Molecules, and Diseases 38 (2007) 120 – 191
signals (mean 0.5 mm) does not suggest direct physical interaction, and combined RNA immunoFISH detecting engaged elongating RNA polymerase II demonstrated that the majority of active associating genes are sitting at different transcription foci. Shared transcription factories are therefore unlikely to be responsible for the observed proximity between genes. In order to understand whether this nuclear positioning is a general phenomenon, we have looked at a panel of human genes up-regulated during erythropoiesis and find that some of these also show increased inter-allelic and inter-genic associations when active, to such a degree that one might expect to observe clusters of several associating genes. By using multicolour FISH, we were able to demonstrate that multiple, upregulated genes group together in the erythroblast nucleus in a common nuclear region. However, we show that this grouping is not due to clustering around common transcription factories. These data support a model of self-assembly in nuclear positioning of genes, rather than a functional requirement for differential gene expression. doi:10.1016/j.bcmd.2006.10.023
13 Dynamic regulation of transcription complex recruitment to the B-globin gene locus Valerie J. Crusselle-Davis, Zhuo Zhou, Shermi Liang, I-Ju Lin, Archana Anantharaman, Vikram Purohit, Tihomir S. Dodev, Felicie Andersen, Jo¨rg Bungert Department of Biochemistry and Molecular Biology, Powell-Gene Therapy Center, Center for Mammalian Genetics, Genetics Institute, University of Florida, Gainesville, FL 32610, USA The human h-globin genes are expressed exclusively in erythroid cells and regulated by a locus control region (LCR) comprised of several DNaseI hypersensitive (HS) sites and located upstream of the globin genes. Our previous work demonstrated that transcription complexes are recruited to individual LCR HS sites prior to appearance at the globin gene promoters during erythroid differentiation of embryonic stem cells. We further showed that RNA polymerase II (RNA Pol II) can be transferred from immobilized LCR templates to globin gene promoters in an in vitro system. We continued these studies and also analyzed transcription complex recruitment to the globin gene locus during and after mitosis. Furthermore, we continued our analysis of the role of transcription factors USF and TFII-I in h-globin gene expression. Our data provide evidence supporting the hypothesis that USF activates h-globin gene expression by recruiting co-activators and histone acetyl transferase activities to the promoter, while TFII-I represses hglobin gene expression by recruiting histone de-acetylases. During erythroid differentiation of embryonic stem cells TFII-I associates with the h-globin promoter at early stages when the gene is inactive. In contrast, the association of USF is accompanied by h-globin gene activation. We currently generate transgenic mice expressing dominant negative
129
mutants of TFII-I and USF in erythroid cells. Data resulting from the analysis of these mice will be presented. doi:10.1016/j.bcmd.2006.10.024
14 DNA looping in the A-globin locus A.Z. Canals-Hamann, J.E. Reittie, V.J. Buckle, F.J. Iborra Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, John Radcliffe Hospital, Headington, Oxford, UK We want to understand the basis of transcription factories. We focused our study in the a-globin locus. Using 3C, we mapped molecular interactions between distant regulatory elements in the a-globin locus. We have found that the promoter region of the a-globin gene interacts with the 26 hypersensitive site in the mouse fetal liver (where alpha-globin is expressed) and not in the brains of the same animals. Interactions between the promoter region of the a-globin gene and other hypersensitive sites such as HS-31, HS-12 and HS-8 were not as prominent, as with HS26. On the other hand, the promoter region of the zeta-globin gene does not seem to be interacting with the distal hypersensitive site and the proximal alpha-globin gene promoter. This is setting up the base for further studies into nuclear organisation, which we will check in combination with FISH. doi:10.1016/j.bcmd.2006.10.025
15 Identification of Zfp148 (ZBP-89) as a novel GATA-1 associated Kruppel-type zinc finger transcription factor required for megakaryopoiesis and definitive erythropoiesis Andrew Woo 1, Tyler B. Moran 1, Seong-Kyu Choe 2, Yocheved Schindler 1, Matthew Sullivan 1, Yuko Fujiwara 1, Barry H. Paw 2, Alan B. Cantor 1 1 Division of Pediatric Hematology-Oncology, Children’s Hospital Boston/Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA 2 Division of Hematology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA The zinc finger transcription factor GATA-1 plays an essential role in megakaryocytic and erythroid terminal maturation. Yet the molecular mechanisms that control GATA-1 function are incompletely understood. Many transcription factors, in addition to binding DNA, make important protein –protein interactions that modulate their activity. Here, we show that GATA-1 participates in stable multiprotein complexes ranging from ¨100 kDa to 700 kDa in murine L8057 megakaryoblastic cells. We generated stable L8057 cell lines expressing metabolically biotinylated and FLAG epitope
signals (mean 0.5 mm) does not suggest direct physical interaction, and combined RNA immunoFISH detecting engaged elongating RNA polymerase II demonstrated that the majority of active associating genes are sitting at different transcription foci. Shared transcription factories are therefore unlikely to be responsible for the observed proximity between genes. In order to understand whether this nuclear positioning is a general phenomenon, we have looked at a panel of human genes up-regulated during erythropoiesis and find that some of these also show increased inter-allelic and inter-genic associations when active, to such a degree that one might expect to observe clusters of several associating genes. By using multicolour FISH, we were able to demonstrate that multiple, upregulated genes group together in the erythroblast nucleus in a common nuclear region. However, we show that this grouping is not due to clustering around common transcription factories. These data support a model of self-assembly in nuclear positioning of genes, rather than a functional requirement for differential gene expression. doi:10.1016/j.bcmd.2006.10.023
13 Dynamic regulation of transcription complex recruitment to the B-globin gene locus Valerie J. Crusselle-Davis, Zhuo Zhou, Shermi Liang, I-Ju Lin, Archana Anantharaman, Vikram Purohit, Tihomir S. Dodev, Felicie Andersen, Jo¨rg Bungert Department of Biochemistry and Molecular Biology, Powell-Gene Therapy Center, Center for Mammalian Genetics, Genetics Institute, University of Florida, Gainesville, FL 32610, USA The human h-globin genes are expressed exclusively in erythroid cells and regulated by a locus control region (LCR) comprised of several DNaseI hypersensitive (HS) sites and located upstream of the globin genes. Our previous work demonstrated that transcription complexes are recruited to individual LCR HS sites prior to appearance at the globin gene promoters during erythroid differentiation of embryonic stem cells. We further showed that RNA polymerase II (RNA Pol II) can be transferred from immobilized LCR templates to globin gene promoters in an in vitro system. We continued these studies and also analyzed transcription complex recruitment to the globin gene locus during and after mitosis. Furthermore, we continued our analysis of the role of transcription factors USF and TFII-I in h-globin gene expression. Our data provide evidence supporting the hypothesis that USF activates h-globin gene expression by recruiting co-activators and histone acetyl transferase activities to the promoter, while TFII-I represses hglobin gene expression by recruiting histone de-acetylases. During erythroid differentiation of embryonic stem cells TFII-I associates with the h-globin promoter at early stages when the gene is inactive. In contrast, the association of USF is accompanied by h-globin gene activation. We currently generate transgenic mice expressing dominant negative
129
mutants of TFII-I and USF in erythroid cells. Data resulting from the analysis of these mice will be presented. doi:10.1016/j.bcmd.2006.10.024
14 DNA looping in the A-globin locus A.Z. Canals-Hamann, J.E. Reittie, V.J. Buckle, F.J. Iborra Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, John Radcliffe Hospital, Headington, Oxford, UK We want to understand the basis of transcription factories. We focused our study in the a-globin locus. Using 3C, we mapped molecular interactions between distant regulatory elements in the a-globin locus. We have found that the promoter region of the a-globin gene interacts with the 26 hypersensitive site in the mouse fetal liver (where alpha-globin is expressed) and not in the brains of the same animals. Interactions between the promoter region of the a-globin gene and other hypersensitive sites such as HS-31, HS-12 and HS-8 were not as prominent, as with HS26. On the other hand, the promoter region of the zeta-globin gene does not seem to be interacting with the distal hypersensitive site and the proximal alpha-globin gene promoter. This is setting up the base for further studies into nuclear organisation, which we will check in combination with FISH. doi:10.1016/j.bcmd.2006.10.025
15 Identification of Zfp148 (ZBP-89) as a novel GATA-1 associated Kruppel-type zinc finger transcription factor required for megakaryopoiesis and definitive erythropoiesis Andrew Woo 1, Tyler B. Moran 1, Seong-Kyu Choe 2, Yocheved Schindler 1, Matthew Sullivan 1, Yuko Fujiwara 1, Barry H. Paw 2, Alan B. Cantor 1 1 Division of Pediatric Hematology-Oncology, Children’s Hospital Boston/Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA 2 Division of Hematology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA The zinc finger transcription factor GATA-1 plays an essential role in megakaryocytic and erythroid terminal maturation. Yet the molecular mechanisms that control GATA-1 function are incompletely understood. Many transcription factors, in addition to binding DNA, make important protein –protein interactions that modulate their activity. Here, we show that GATA-1 participates in stable multiprotein complexes ranging from ¨100 kDa to 700 kDa in murine L8057 megakaryoblastic cells. We generated stable L8057 cell lines expressing metabolically biotinylated and FLAG epitope